The present invention relates to a method for the base sequencing of deoxyribonucleic acid (DNA). More specifically, this invention relates to methods of sequencing both the sense and antisense strands of DNA through the use of blocked and unblocked sequencing primers.
Genome sequencing offers the possibility of diagnosis, therapy and prevention of illnesses as well as the targeted modification of the human genome. Rapid sequencing methods are required to allow the use of this potential. Base sequencing of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) is one of the most important analytical techniques in biotechnology, the pharmaceutical industry, food industry, medical diagnostics and other fields of application.
There are many DNA sequencing methods available, such as the Sanger sequencing using dideoxy termination and denaturing gel electrophoresis (Sanger, F. et al., Proc. Natl. Acad. Sci. U.S.A. 75, 5463-5467 (1977)), Maxam-Gilbert sequencing using chemical cleavage and denaturing gel electrophoresis (Maxam, A. M. & Gilbert, W. Proc Natl Acad Sci USA 74, 560-564 (1977)), pyro-sequencing detection pyrophosphate (PPi) released during the DNA polymerase reaction (Ronaghi, M. et al., Science 281, 363, 365 (1998)), and sequencing by hybridization (SBH) using oligonucleotides (Lysov, I. et al., Dokl Akad Nauk SSSR 303, 1508-1511 (1988); Bains W. & Smith G. C. J. Theor Biol 135, 303-307(1988); Drnanac, R. et al., Genomics 4, 114-128 (1989); Khrapko, K. R. et al., FEBS Lett 256. 118-122 (1989); Pevzner P. A. J Biomol Struct Dyn 7, 63-73 (1989); Southern, E. M. et al., Genomics 13, 1008-1017 (1992)).
Ronaghi et al., (Anal. Biochem. 267, pp. 65-71 (1999)) referred to a method of sequencing both strands of a nucleic acid. The method involves PCR amplification of a template nucleic acid with a biotinylated primer and a non-biotinylated primer. The amplified product, comprising a biotinylated strand and a non-biotinylated strand is strand separated. The authors referred to the use of streptavidin coated bead for strand separation. The biotinylated strand remains attached to the bead while the non-biotinylated strand is separated from the bead under denaturing conditions. The two strands (biotinylated and non-biotinylated) are sequenced separately. Unlike the present invention which uses solid-phase sequencing for both strands, this method uses solid-phase sequencing for the biotinylated strand and solution phase sequencing for the non-biotinylated strand. Hence, Ronaghi's method is similar to the traditional method of DNA sequencing comprising strand separation (e.g., using a urea gel) of a template before sequencing. Thus, Ronaghi's method suffers from the same disadvantage as the traditional methods; requirement of a labor intensive step of strand separation and isolation of individual strands prior to sequencing. The disadvantages of Ronaghi's method increases geometrically as the number of parallel sequencing reactions increase. For example, the parallel sequencing of 1000 double stranded templates would require 1000 separations and 2000 single strand isolations. Furthermore, Ronaghi's method, like all methods based on strand separation, is limited to the determination of sequences from two primers (one for each strand) per double strand template.
Sequencing based on chemical cleavage has proven to be difficult to automate. Other sequencing methods are labor intensive due to the need to perform a hybridization step for every sequencing effort. In many situations, the hybridization step is the rate-limiting step in a sequencing reaction.
Attempts have been made for sequencing from two ends of a nucleic acid using, for example, two distinctly labeled sequencing primers (e.g., Li-Cor of Lincoln, Nebraska) in a Sanger sequencing reaction (Sanger, F. et al., Proc. Natl. Acad. Sci. U.S.A. 75, 5463-5467 (1977). These methods are variations of manual sequencing and require a size fractionation (e.g., a gel) step to determine a sequence. While size fractionation may be suitable for small sample sizes, genomic sequencing using size fractionation techniques would require 1,500,000 size fractionation gels (assuming an optimistic capability of 2000 bp per gel). For these reasons, sequencing methods involving size fractionation have not been adapted to sequencing of human genomes.